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D L Richardson

Publications and source records attributed to D L Richardson.

34 records · Page 2Linked to original sources

Progression of an inductive signal activates sporulation in Dictyostelium discoideum.

spiA, a marker for sporulation, is expressed during the culmination stage of Dictyostelium development, when the mass of prespore cells has moved partly up the newly formed stalk. Strains containing a full-length spiA promoter/lacZ fusion were stained for beta-galactosidase activity at intervals during development. The results indicate that expression of spiA initiates in prespore cells at the prestalk/prespore boundary (near the apex) and extends downward into the prespore mass as culmination continues. A spatial gradient of staining expands from the top of the prespore mass and intensifies until the front of activation reaches the bottom, whereupon the entire region stains darkly. The spiA promoter can be deleted to within 301 bp of the transcriptional start site with no effect on the relative strength, timing or spatial localization of expression. Further 5' deletions from -301 to -175 reduce promoter strength incrementally, although timing and spatial expression are not affected. Deletions to -159 and beyond result in inactive promoters. Treatment of early developmental structures with 8-Br-cAMP in situ activates the intracellular cAMP-dependent protein kinase (PKA) and precociously induces spiA expression and sporulation. The absence of an apparent gradient of staining in these structures suggest that PKA is equivalently activatable throughout the prespore region and that all prespore cells are competent to express spiA. Thus, we postulate that the pattern of expression of spiA reveals the progression of an inductive signal for sporulation and suggest that this signal may originate from the prestalk cells at the apex.

8-Bromo Cyclic Adenosine Monophosphate↗

Disruption of the sporulation-specific gene spiA in Dictyostelium discoideum leads to spore instability.

The spiA gene of Dictyostelium is expressed specifically in prespore cells and spores during culmination, the final stage of development during which prespore and prestalk cells undergo terminal differentiation to form spores and stalk. We have used homologous recombination to delete this gene and have characterized the resulting phenotype. The spiA- strains develop normally and produce spores that are indistinguishable from those of wild-type strains by transmission and scanning electron microscopy. Mutant spores have normal viability when assayed soon after the completion of development, but, as the spiA- spores age, they lose viability more rapidly than those of the spiA+ parent. The drop in viability is more pronounced when spores are submerged in dilute buffer at a concentration that does not allow germination; after 11 days submerged, the viability of spiA- spores is 10(5)-fold reduced, whereas that of the parent is decreased only 10-fold. Reinserting an intact copy of the spiA gene into a spiA- strain restores the stability of its spores. The product of the spiA gene, Dd31, was identified on Western blots as a 30-kD protein using an antibody raised against a fusion protein containing a portion of the coding sequence. Dd31 is associated with the inner face of spore coat fragments in a detergent-resistant manner. This location is consistent with its observed role in maintaining stability of the spores.

Animals↗

A prespore gene, Dd31, expressed during culmination of Dictyostelium discoideum.

During culmination of Dictyostelium fruiting bodies, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. A genomic fragment was isolated by random cloning that hybridizes to a 1.4-kb mRNA present during culmination. Cell type separations at culmination showed that the mRNA is present in prespore cells and spores, but not in prestalk or stalk cells. After genomic mapping, an additional 3 kb of DNA surrounding the original 1-kb fragment was cloned. The gene was sequenced and named Dd31 after the size of the predicted protein product in kilodaltons. Accumulation of Dd31 mRNA occurs immediately prior to sporulation. Addition of 20 mM 8-Br-cAMP to cells dissociated from Mexican hat stage culminants induced sporulation and the accumulation of Dd31 mRNA, while 20 mM cAMP did not. Dd31 mRNA does not accumulate in the homeotic mutant stalky in which prespore cells are converted to stalk cells rather than spores. Characterization of Dd31 extends the known temporal dependent sequence of molecular differentiations to sporulation.

Amino Acid Sequence↗

Proteolysis of bacteriophage phi X174 prohead protein gpB by a protease located in the Escherichia coli outer membrane.

The gene B protein (gpB) of bacteriophage phi X174 is required for prohead assembly and is removed from prohead during phage maturation. Protease activity was observed in isolated prohead which specifically cleaved gpB. Cleavage of gpB produced two fragments that had apparent molecular weights of 12,300 and 3,700 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequencing of the fragments confirmed that they resulted from the cleavage of gpB and identified the cleavage site as an Arg-Arg at amino acids 76 to 77 of the 120-amino-acid protein. gpB-specific protease activity was observed in both phi X174-infected and uninfected Escherichia coli extracts. This protease activity was localized to the outer-membrane fraction of uninfected cells. Protease activities present in the outer membrane and in isolated prohead produced identical fragments and had the same protease inhibition profile. The gpB-specific activity in uninfected cells was induced by growth at 42 degrees C and was inhibited by the protease inhibitors, 1,10-phenanthroline, EDTA, and N-ethylmaleimide.

Amino Acid Sequence↗

Neutrophil accumulation in vivo following the administration of chemotactic factors.

Intravenously administered 51Cr-labelled homologous neutrophils accumulated at guinea-pig skin sites prepared by intradermal injection of factors derived from complement-activated serum, possessing in vitro chemotactic activity. There was a strong correlation between the in vitro potency of Sephadex G-100 fractions of the activated serum and their ability to evoke neutrophil accumulation in vivo. These experiments suggest that agents which are chemotactic for neutrophils in vitro also induce the localization of this cell in vivo.

Animals↗

Chemotaxis for human monocytes by fibrinogen-derived peptides.

Chemotactic activity for human peripheral blood monocytes was generated by the action of thrombin on human fibrinogen, a reaction known to release low molecular fibrinopeptides. Supernatants prepared by incubating Contortrix venom with fibrinogen, which cleaves the B peptide, were also chemotactic, whereas no activity was present in supernatants from Arvin venom, which cleaves peptides A, AP and AY. Thus fibrinopeptide B was chemotactic for the monocyte in addition to the neutrophil, as previously reported. Monocyte chemotactic activity was also generated by the action of plasmin on human fibrinogen and shown to be associated with the D and E fragments but not with a mixture of fragments X and Y. When plasmin digestion was stopped at time intervals up to 24 h, monocyte chemotactic activity corresponded with the appearance of the D and E fragments. The monocyte chemotactic activity, contained in a 24 h digest, eluted from Sephadex G-75 at VO, corresponding to the expected position of the D and E fragments whereas neutrophil chemotactic activity eluted with molecules of molecular size of approximately 30 000 daltons. Thus fragments D and E derived from plasmin digestion of fibrinogen attract the monocyte whereas only the small uncharacterized peptides were chemotactic for the neutrophil. These different profiles of chemotactic activity for the neutrophil and the monocyte in terms of plasmin digestion products of fibrinogen may be of significance in the events leading to the accumulation of these cells in vivo during fibrin deposition.

Chemotaxis↗

Combined percutaneous and endoscopic procedures for bile duct obstruction: simultaneous and delayed techniques compared.

BACKGROUND/AIMS: In patients with obstructive jaundice, when the endoscopic approach fails to achieve biliary drainage, percutaneous cannulation and combined endoscopic/percutaneous endoprosthesis insertion can be performed simultaneously or in stages. This study compared these two approaches. METHODOLOGY: Over a three-year period 41 patients were studied. All had obstructive jaundice for which endoscopic drainage had failed. In 22 patients (group 1) percutaneous transhepatic drainage was followed a few days later by combined endoscopic and percutaneous procedure. In 19 patients (group 2) the percutaneous transhepatic drainage and combined drainage were performed at the same session. In the multiple stage group the mean interval between the first endoscopic retrograde cholangiopancreatography and final combined procedure was 9 days (SD 5.2). The groups were similar for sex, underlying pathology and reasons for failure of endoscopic approach. Group 1 patients were older 73 vs. 65 years (p < 0.05). RESULTS: Patients in group 2 had a more rapid recovery and discharge home: mean 6 days, compared to mean 18 days from the initial procedure for group 1 (p < 0.001). Five patients died of their disease without leaving hospital (4 in group 1, 1 in group 2). In each group drainage failed in 1 patient. Complications were more common in group 1: 73% vs. 37% (p < 0.05). Pancreatitis (3 vs. 2) and septicemia (4 in group 1, 3 in group 2) were similar but group 1 had complications from the external drain: cholangitis and pyrexia in 4 patients, 3 bile leaks, and 1 catheter displacement. CONCLUSIONS: When endoscopic drainage alone fails, a combined percutaneous/endoscopic procedure should only be performed if it can be carried out simultaneously.

Aged↗